It is well-known that the expression of genes can be modulated by the effects of the DNA sequence surrounding the gene. One such method is the insertion of a sequence which, when transcribed to RNA, forms a self-cleaving ribozyme (see, e.g., Prody et al Science 231: 1577-1580 (1986)).
Programmable RNA-based gene-regulatory devices have been designed with functional RNA parts that encode sensing, information transmitting, and actuating functions (Win et al Proc. Natl. Acad. Sci. 2007 104: 14283-8). RNA device architectures functionally connect sensor and actuator components, such that sensor-detected information is transmitted into controlled activity of the actuator domain. One class of RNA devices utilizes a hammerhead ribozyme (HHRz) actuator to modulate the stability of a target transcript through conditional control of ribozyme cleavage activity via binding of the cognate ligand. The ribozyme-based device framework supports the design of robust genetic controllers in different organisms, responsive to diverse ligands, exhibiting complex computation, and applied to regulate complex phenotypes. Typical design strategies link sensor and actuator components through a rationally designed or screened transmitter component that guides secondary structure conformation changes of the functional components.
However, these methods require a sensor that not only detects the molecule of interest but also functions correctly in the context of the device, effectively converting the concentrations of the molecules into control of the actuator domain. Existing work in finding aptamers that function as sensors typically use aptamers found using methods based on binding. These do not provide the sensor in the context of the device and often require chemical modifications of the target of interest. Other techniques that use a SELEX-based process to find aptamers that function as sensors in the context of an otherwise fixed ribozyme-based device, such as “allosteric selection” (Koizumi et al Nat. Struct. Biol. 1999 6: 1062-1071; Soukup et al, J. Mol. Biol. 2000 298: 623-632), have had limited success and require labor-intensive steps in each round that are not amenable to automation. Coupled with the need for many rounds of selection to isolate desirable sensor sequences from undesired amplicons, prior processes have limited practical utility.